This project examines the immunoregulatory functions of a T-cell factor and a macrophage factor (MCDF, MO-derived cytotoxic T-cell differentiation factor) that are involved in the generation of cytotoxic T lymphocytes (CTL). Both factors are distinct from interleukin 1, interleukin 2, and interferon. The T-cell factor, which coelutes with colony-stimulating factor (CSF), functions in CTL development by inducing MO to produce a CTL activation factor. MCDF appears to act on CTLs to provide an activation/differentiation signal that syngerizes with IL-2 for the generation of a cytotoxic response. MCDF generates CTLs from Lyt 2+ responder cells in mixed lymphocyte cultures (MLC). Recently, we used CTL clones to define the activation/differentiation function of MCDF and other factors in the induction of a cytotoxic response. MCDF restores cytotoxic activity to a CTL clone (B6D/2-7c obtained from C. Orosz, Columbus, OH) that lost its cytolytic capacity following treatment with phorbol myristate acetate (PMA). This restoration of cytolytic activity to PMA-treated B6D/2-7c cells was independent of cell proliferation. In addition, MCDF augmented the cytolytic activity of untreated B6D/2-7c cells, suggesting that MCDF can provide a signal to CTL clones that is not dependent on PMA treatment. Neither IL-1, IL-3, nor CSF restored the cytolytic activity to PMA-treated B6D/2-7c cells. However, IL-2 induced a cytotoxic response from PMA-treated B6D/2-7c cells even in the presence of cytosine arabinoside. The ability of IL-2 to induce a cytotoxic response from inactivated B6D/2-7c cells was not attributable to IL-2 inducing interferon. Studies are in progress to: (1)\characterize and purify the T-cell factor and MCDF further; (2)\identify their mechanism of action; and (3)\determine their relationship to other immunoregulatory molecules. (HF)